Research focused on investigating why narcolepsy patients tend to be overweight—despite the fact that they eat less than normal—has uncovered a potential new target for the treatment of obesity. Investigators at Sanford-Burnham Medical Research Institute have found that obesity in mice lacking the neuropeptide orexin (OX) is associated with a deficiency in brown adipose tissue (BAT) thermogenesis that results from the inability of brown preadipocytes to differentiate, ramp up respiration, and accumulate triglycerides.

OX deficiency is also observed in human narcolepsy patients, and the new studies in mice suggested that the lack of OX results in brown fat hypoactivity that effectively leads to dampening of energy. The adipocyte differentiation defect in OX-null neonates was prevented by giving their pregnant mothers OX injections, an approach that triggered a full differentiation program in mesenchymal progenitor stem cells, embryonic fibroblasts, and brown preadipocytes. Devanjan Sikder, Ph.D., Dyan Sellayah, Ph.D., and Preeti Bharaj, Ph.D., describe their work in Cell Metabolism in a paper titled “Orexin Is Required for Brown Adipose Tissue Development, Differentiation, and Function.”

Orexin A and B are multifunctional neuropeptides that bind to orexin receptors OX1 and OX2 to regulate sleep-wake cycles, physical activity, and appetite. Dscovery of OX deficiency in human narcolepsy was first reported back in 2000, the Sanford-Burnham team explains.

An association between obesity and narcolepsy, meanwhile, was first made some 80 years ago, and since then studies have demonstrated that both humans and mice who are deficient in OX develop obesity even if they eat much less than normal individuals. The fact that narcoleptic patients are more obese than equally active subjects with idiopathic hypersomnia (excessive sleeping with no obvioius cause), also strongly indicates that orexin deficiency in narcoleptic patients is involved in the pathology underlying the disease. Yet despite these pointers, the pathogenesis of obesity in narcolepsy remains unclear.

To study the mechanisms involved in the development of obesity, the researchers turned again to OX-null mice. They initially observed that the OX-deficient animals rapidly gained far more weight than control animals when fed a high-fat diet (HFD), but not when fed a regular chow diet, indicating that lack of OX is more likely to cause obesity in conditions of calorific excess. This excess weight gain by OX-null mice fed a HFD occurred even though they tended to eat less than wild-type animals, and displayed no difference in terms of physical activity or energy expenditure. Rather, the HFD-fed OX-deficient animals gained more weight per gram of food consumed.

This absence of diet-induced energy expenditure and O2 consumption in OX knockout (KO) mice prompted them to look at the animals’ BAT function. Genetic analysis of intrascapular BAT (iBAT) collected from chow and HFD-fed mice showed that the OX-deficient animals’ failure to increase metabolic rate in response to diet was accompanied by reduced of BAT-related thermogenic genes known to be induced by HFD. This suggested that there might be an underlying defect in brown-fat activity that caused metabolism-related changes impacting on DIT, and causing obesity.

Direct comparison of iBAT from wild-type and OX-null chow-fed mice showed that while there was no difference in tissue size and weight, it did appear paler, and lacked the dense clusters of lipid droplets evident in the iBAT of wild-type mice. Moreover, all the OX-deficient animals demonstrated significant reduction in intracellular triglycerides, amounting to 88% less than in wild-type animals, while the expression of key factors involved in brown-fat differentiation and function was also reduced. “Pronounced reduction in iBAT lipid stores in the face of impaired Ppar-γ1, Ppar-γ2, Pgc-1α, Pgc-1β, and Ucp-1 mRNA expression suggest that brown adipocytes are incapable of efficiently synthesizing triglycerides,” the authors write. This, they postulated, may be due to the failure of brown preadipocytes to differentiate in the absence of OX.

In rodents, BAT develops during fetal life, continues until 3–5 days after birth, and is most concentrated in the interscapular region. In contrast, white fat develops after birth. In neonates, BAT comprises cells at distinct stages of differentiation: preadipocytes demonstrate no cytoplasmic lipid accumulation, but these differentiate into immature adipocytes that can accumulate small cytoplasmic lipid droplets, and the immature adipocytes then further differentiate into mature adipocytes containing well-developed lipid droplets.

Comparison of iBAT from wild-type and OX-null neonates showed no significant differences in terms of tissue development, but fat droplet abundance, size, and mitochondrial content were drastically reduced in OX-null newborn iBAT. Expression of the transcriptional regulators Ppar-γ1, Pgc-1α, and Pgc-1β was markedly reduced, “suggesting that differentiation in majority of iBAT cells in OX-null mice may be arrested in the preadipocyte stage,” the team notes.

Interestingly, evaluation of neonatal or adult BAT mRNA in wild-type animals failed to detect OX expression, but moderate levels of OX mRNA were produced in the placenta. The team therefore reasoned that “if OX is physiologically required for BAT development, OX therapy during prenatal growth might rescue the BAT defect in OX-null pups.”

Pregnant OX-null mice were given three intraperitoneal injections of OX during the course of their pregnancies, and iBAT from OX-null newborns was analyzed. BAT from these animals demonstrated a marked improvement in iBAT morphology, and an increase in mitochondrial content, indicating that OX treatment of pregnant knockout animals supported BAT differentiation in the OX-null pups. The improvements were accompanied by near-normal mRNA expression of PPpar-γ1, Pgc-1α, and Pgc-1β. “These observations unravel a previously unrecognized role of OX in embryonic development of BAT,” the authors note.

The findings thus far hinted that OX might directly bind to OX receptors on mesenchymal stem cells (MSCs) to trigger brown adipocyte lineage commitment during fetal development. The researchers looked into this further using cultured C3H10T1/2 MSCs, a cell lineage capable of differentiating into fibroblastic, myogenic, adipogenic, osteogenic, and chondrogenic lineages. Initial tests showed that the undifferentiated cells expressed the OX receptor OXR1, but didn’t express OX itself. However, culturing C3H10T1/2 MSCs in OX prior to switching to a standard differentiation medium led to equivalent levels of cytoplasmic triglyceride accumulation as in MSCs treated with BMP-7, a known inducer of BAT differentiation.

Further evaluation showed that 90% of MSCs treated with OX took on a morphology characteristic of differentiated fat cells, and the adipocytes contained multiple oil droplets and demonstrated extensive mitochondrial biogenesis. The early transcriptional regulators of adipogenesis, PRDM16, PGC-1γ, PPAR-γ1, and C/EBP-α, were also elevated both at the mRNA and protein levels, as were mRNA and protein expression of the terminal BAT marker, UCP-1, and genes involved in mitochondrial biogenesis and function. OX treatment of MSCs also suppressed mRNA expression of all three adipogenic inhibitory factors that are typically expressed in undifferentiated MSCs.

OX-differentiated C3H10T1/2 cells consequently demonstrated higher respiration than control cells, and 50% of the elevated respiration was uncoupled from ATP synthesis, which is an attribute of brown fat, the authors remark.

Significantly, treatment of HIB1B cells—a well-established cell line for brown preadipocytes—with OX for just 24 hours induced extensive lipid accumulation without the need for other the adipogenic factors, and OX also induced differentiation of primary brown adipocytes isolated from 1-day-old wild-type C57BL6 mice, and induced differentiation of mouse embryonic fibroblasts to a BAT lineage. Differentiation of C3H10T1/2 cells into adipocytes could also follow transfection with an OX-overexpressing lentivirus, instead of culturing the cells with exogenous OX.

The assumption that OX binds to OXR1 expressed by adipocyte precursor cells was confirmed through experiments using a short hairpin RNA (shRNA) targeting the OX receptor, or treatment with a selective OXR1 antagonist. In these cells, treatment with exogenous OX failed to trigger differentiation and blunted OX-induced lipid accumulation and mitochondrial biogenesis. Primary brown preadipocytes isolated from OXR1-knockout mice were similarly far less capable of differentiating into adipocytes, even when treated with orexin. However, cultured in the presence of a standard adipogenic cocktail, OXR1-null preadipocytes differentiated just as efficiently as wild-type preadipocytes. “This suggests that OXR1 signal activation is not required for hormone-assisted brown-fat differentiation,” the team writes.

The results and broad conclusions drawn from OX-null narcoleptic mouse model and in vitro cell culture is relevant to the understanding of energy expenditure regulation and in the control of obesity in general, the authors conclude. “A plethora of reports show a strong association between low plasma OX and obesity in humans. Though it’s difficult to extrapolate animal studies into humans, findings reported here predict that the sensitivity to obesity in OX deficiency could be primary to hypoactive BAT...Increasing energy expenditure by enhancing BAT function through differentiation would be a viable approach to prevent or reduce obesity. This warrants future studies investigating the potential of OX or OXR1 agonists in improving BAT function and triggering weight loss.”